Abstract
Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis in the hippocampus, inducing learning-memory deficits; however, underlying mechanisms remain less understood. Here, we explored the molecular mechanism in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and growth factor signaling that relate with apoptosis. We observed enhanced hippocampal autophagy in OVX rats, characterized by increased levels of autophagy proteins, presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17β-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with ERα and memory performance, disrupted in estrogen-deficient condition.
Introduction
The primary female sex hormone, estrogen, activates its receptors, ERα and ERβ, and exhibits physiological functions within the brain (Mukai et al. 2010, Hara et al. 2015). Correspondingly, ovarian failure and estrogen deficiency, which characterize menopause in women, suppress cerebral ER levels (Qu et al. 2013, Fang et al. 2018) and alter neuronal functions, including cognition (Kim et al. 2016, Djiogue et al. 2018). Estrogen deficiency induces hippocampal apoptosis, neuronal loss (Sales et al. 2010, Yazgan & Naziroglu 2017) and cognitive dysfunctions (Kim et al. 2016, Djiogue et al. 2018), while estradiol therapy reduces apoptosis and improves learning-memory performances (Sales et al. 2010, Uzum et al. 2016, Yazgan & Naziroglu 2017). However, the underlying mechanisms remain obscure.
The biological self-degradative phenomenon, autophagy, sustains cell survival and plays an essential role in CNS homeostasis (Cheung & Ip 2011). Although autophagy helps to remove denatured proteins and damaged organelles, unrestrained autophagy activation prompts undue neuronal self-digestion and cognitive deficits (Wong & Cuervo 2010, Li et al. 2014). Accordingly, deregulated neuronal expression of autophagy regulators, mammalian target of rapamycin (mTOR) and Unc-51 like autophagy-activating kinase (ULK1), autophagy-related proteins, ATG7 and ATG5/12, microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1 triggers cognitive decline (Herrera et al. 2008, Yu et al. 2017). Additionally, an altered expression of mitochondrial DNA-encoded genes, cytochrome B (CytB) and cytochrome c oxidase subunit II (COXII), and mitochondrial markers, voltage-dependent anion channel 1 (VDAC1) and COXIV, together with selective autophagic mitochondria removal, that is, mitophagy, contributed toward impaired neuronal functions (Lu et al. 2014, Shaerzadeh et al. 2014). A PTEN-induced kinase 1 (PINK1)/Parkin pathway is also important for regulating autophagy-mediated mitochondrial clearance in the neurons (Amadoro et al. 2014). Moreover, although less studied in neurons, the mitochondrial BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1) played a key role in regulating mitophagy (Lampert et al. 2019).
A complex cross-talk between apoptosis and autophagy has also been reported, where enhanced autophagy ameliorated cognitive deficits through activation of hippocampal neuronal apoptosis (Li et al. 2016). Contrarily, reduced ATG5 levels via attenuated caspase-mediated apoptosis appeared neuroprotective (Zhang et al. 2016a , b ). Nonetheless, whether estrogen deficiency had any impact on hippocampal neuronal autophagy and whether the latter related to apoptosis, cognitive impairments or had any association with mitochondria awaits investigation.
The intersection between growth factors, apoptosis and autophagy serves as an attractive target for disease intervention within the brain (Garcia-Huerta et al. 2016). Growth factor signal transduction pathways often stimulate PI3K-AKT-mTOR mechanism and inhibit neuronal autophagy (Kalkman & Feuerbach 2017). Although primarily studied in glial cells, epidermal growth factor receptor (EGFR) signaling constitutes one such pathway that controls autophagy (Ghildiyal et al. 2013). Dysregulated EGFR signaling inactivates Beclin-1, reduces endogenous LC3II and p62 levels and activates PI3K/AKT/mTOR cascade for suppressing glial lethality (Palumbo et al. 2014). In fact, combined EGFR-autophagy modulation has been envisaged as a therapeutic strategy for reduced glioblastoma cell invasiveness and improved neurological performances (Palumbo et al. 2014, Tini et al. 2015). However, co-existence of neuronal EGFR and autophagy signaling pathways, particularly within the hippocampus, is unknown.
The key EGFR ligands include EGF, heparin-binding epidermal growth factor (HB-EGF), transforming growth factor alpha (TGFα) and betacellulin, and their genetic ablation or reduced expression within the hippocampal neurons caused psychomotor impairments and cognitive dysfunctions (Sasaki et al. 2015, Maurya et al. 2016). EGFR regulates estradiol-mediated functions in the brain, with reports suggesting participation of TGFα and EGF, particularly in the astroglial and neural stem cells (Brannvall et al. 2002, Lee et al. 2012). However, whether any link existed between estrogen and EGFR signaling within the hippocampal neurons remains un-elucidated.
By using ovariectomy (OVX) model of estrogen loss, supported by in vitro studies, we focused on the effect of estrogen deficiency on autophagy, mitochondrial content and EGFR signaling and their link with hippocampal neuronal apoptosis and cognition. Overall, our study identifies a novel estradiol-induced neuroprotective mechanism that sustains hippocampal neuronal cell survival and cognitive functions.
Materials and methods
Reagents
Cell lytic reagents, In Situ Cell Death Detection Kit Fluorescein (ref. no. 11684795910), Bradford reagent and 17β-estradiol (cat no. E8875) were procured from Sigma. Immobilon Western chemiluminescent HRP Substrate and 3-Methyladenine (3-MA; cat no. 189490) were from Millipore. Lipofectamine 2000 transfection reagent, non-targeting (NT)-siRNA (cat no. AM4611), Becn1-siRNA (Assay ID s137745), Esr1-siRNA (Assay ID s128616) and MitoTracker™ Red CMXRos (cat no. M7512) were from Invitrogen. Superscript™ III First-Strand Synthesis kit (cat no. 18080051) and Maxima SYBR Green/ROX qPCR master mix (2×) were from Thermo Fischer Scientific. Wizard® Genomic DNA Purification Kit (A1120) was from Promega. Recombinant HB-EGF was from R&D systems. Vectashield Antifade Mounting Medium with DAPI was from Vector Laboratories. Tandem-tagged mt-RFP EGFP plasmid was a kind gift from Dr Andreas Till (Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany). Serum estradiol (E2) kit (cat no. E-EL-0065) was from Elabscience (Houston, TX, USA).
Antibodies
Rabbit polyclonal ERα, goat polyclonal HB-EGF and horseradish peroxidase (HRP)-conjugated mouse monoclonal β-Actin were from Santa Cruz Biotechnology. Rabbit polyclonal LC3B, phospho-Akt, Akt and phospho-ULK1 and rabbit monoclonal phospho-AMPKα, AMPKα, ULK1, phospho-EGFR, EGFR, ATG-5/12 conjugate, VDAC1, COXIV, PINK1, Parkin, cleaved caspase-3 and PARP were from Cell Signaling Technology. Rabbit monoclonal phospho-mTOR and rabbit polyclonal mTOR were from Abcam. Mouse monoclonal, Neuronal Nuclei, NeuN, was from Millipore. Rabbit polyclonal Beclin-1, ATG-7 and p62/SQSTM1, and HRP-conjugated secondary anti-mouse, anti-rabbit and anti-goat antibodies were from Sigma. Alexa Fluor®546 goat anti-rabbit IgG, Alexa Fluor®488 goat anti-mouse IgG and Alexa Flour®594 chicken anti-goat IgG were from Invitrogen.
Animal ethical approval
Wistar rats were used after obtaining approval, specific to this study, from the Institutional Animal Ethics Committee of CSIR-CDRI and CSIR-IITR. For maintenance and handling of the rats, guidelines and regulations of the ethics committee were strictly followed.
Animal treatment, surgery and hippocampal tissue isolation
For OVX surgery, adult female rats (250–300 g) were kept in a hygienic condition in well ventilated cages at 24 ± 2°C, 40–60% humidity and 12-h light–darkness cycle with ad libitum availability of diet and water. The rats were subjected to sham- and OVX (bilateral)-surgery following our previously described protocol (Sharan et al. 2011). Briefly, rats were anesthetized with ketamine and xylazine (intraperitoneally, 60 and 20 mg/kg body weight, respectively). An incision was then made at the abdominal wall, adipose tissues cleaned and ovaries removed (OVX). The sham-operated rats underwent same surgical procedure excepting ovary removal (Sham). Rats were injected with gentamicin (50 mg/kg, intramuscular for 3 consecutive days) to minimize post-surgical infection. To determine the effect of 17β-estradiol (E2; 100 µg/kg/day), an earlier protocol with slight modifications was followed (Yazgan & Naziroglu 2017), where the rats were treated subcutaneously for fourteen days from seventh day post OVX. To determine the effect of HB-EGF and autophagy, HB-EGF (100 ng in 2 µL sterile saline) and the autophagy inhibitor, 3-MA (300 nM in DMSO) were injected into the hippocampus once on the 14th day post-OVX surgery, following the protocol described before (Pandey et al. 2017). Tissue samples were collected on the 21st day after OVX surgery, following earlier studies showing hippocampal damage at this time point (Sales et al. 2010, Yazgan & Naziroglu 2017), and supported by our selection criteria of autophagy (showing significantly increased LC3-II) (Fig. 1A). For isolating samples for Western blotting, rats were killed by cervical dislocation and brain dissected out. Hippocampal tissues were isolated, quickly snap frozen in liquid nitrogen, stored in −80°C and processed for Western blotting. For immunohistochemistry (IHC), rats were anesthetized, transcardially perfused using 4% PFA and 0.2% picric acid in phosphate-buffered saline and the whole brains were taken, as described before (Pandey et al. 2017). Blinding and randomization were not carried out for the study. No sample size calculation through statistically methods was performed. However, there were no sample size differences between the beginning and end of the experiments.
OVX induces hippocampal autophagy. Hippocampal tissues were isolated at the 14th, 21st and 42nd day post sham- and -OVX surgery of rats. (A) Representative Western blot and densitometry of LC3-II normalized with β-actin. Hippocampal tissues from Sham, OVX and OVX + E2-treated rats were isolated at the 21st day post surgery. (B and D) Representative Western blot and densitometry of autophagy markers (B) and autophagy regulators (D) normalized with β-actin (B) and respective non-phospho counterparts (D). (C) qPCR analysis showing mRNA levels of P62 normalized with housekeeping gene, Gapdh. (E) Electron micrographs, and inset for OVX, showing autophagosome. Data are representatives of three rats/group and means ± s.e. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to sham or as indicated.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Serum estradiol assay
Blood was collected from rats, centrifuged at 1500 g for 10 min and serum was isolated. Serum E2 was measured using an ELISA assay kit, following the manufacturer’s protocol, and expressed as pg/mL.
Primary neuronal culture
Primary hippocampal neurons were cultured from embryonic-16 (E16) rats, as described earlier (Pandey et al. 2017). Briefly, the hippocampus was isolated in Hank’s Balanced Salt Solution (HBSS), meninges removed, followed by mechanical and enzymatic (0.05% trypsin-EDTA) digestion. The hippocampal suspension was then centrifuged (2000 g , 10 min). The pellet obtained was suspended in neurobasal medium and streptomycin (100μg/mL) and penicillin (100 units/mL), supplemented with N2 (1%), B-27 (2%) and l-glutamine (2mM) (complete medium) and seeded onto Poly-l-Lysine (PLL)-coated culture flasks.
Protein extraction and Western blotting
Hippocampal tissues and neurons were homogenized in lysis buffer containing dithiothreitol (1 mM) and protease inhibitor cocktail, in addition to sodium-orthovanadate and sodium fluoride for phosphorylated proteins. The lysates were then centrifuged (20,000 g , 30 min, 4°C). For mitochondrial protein-based studies, we isolated crude mitochondria from hippocampal tissues through differential centrifugation following an earlier protocol (Dixit et al. 2013). Protein content in supernatant was estimated using Bradford reagent, and SDS-PAGE (4–15%) performed with the samples (50 μg), as described previously (Pandey et al. 2017). Following transfer onto PVDF membrane, blots were probed with LC3B, Beclin-1, ATG-7, ATG-5/12, p62, p-Akt, Akt, p-mTOR, mTOR, p-AMPKα, AMPKα, p-ULK1, ULK1, HB-EGF, p-EGFR, EGFR, VDAC1, COXIV, PINK1, PARKIN, cleaved caspase-3, PARP (1:1000, overnight) and β-actin (1:5000, 2 h) primary antibodies. Blots were then washed with TBST (Tris buffer pH 7.4 (10 mM Tris and 150 mM NaCl) with 0.01% Tween 20)) and incubated with HRP-conjugated secondary antibodies at double the primary antibody dilution. Protein bands were detected with Immobilon Western Chemiluminescent HRP Substrate in an Amersham Imager 600 system (GE Healthcare Life Sciences) and quantified using image analyzer software Quantity One (Bio-Rad).
Transmission electron microscopy (TEM)
Following perfusion-fixation of the rats in PFA and glutaraldehyde, hippocampal tissues were post-fixed in 1% osmium-tetroxide, dehydrated using acetone series (15–100%) and embedded in araldite-dodecenyl succinic anhydride mixture (Ladd Research Industries, Burlington, VT, USA). After baking (60°C), the sections were cut (60–80 nm) using Ultra-microtome (Leica) and picked onto 200-mesh copper grids. Sections were double-stained with uranyl-acetate and lead-citrate and observed under FEI Tecnai G2 spirit twin transmission electron microscope equipped with Gatan digital CCD camera (Pleasanton, CA, USA) at 80 KV as described before (Rai et al. 2013).
Real-time PCR (quantitative PCR, qPCR )
RNA was isolated from the hippocampal tissue using TRIzol reagent and subjected to cDNA synthesis using reverse transcription PCR (Superscript III, Life Technologies), as described before (Pandey et al. 2017). The synthesized cDNA was subjected to qPCR using SYBR green dye and primers (Table 1) (Integrated DNA Technologies, Coralville, IA, USA) over sequential reaction comprising denaturation (95°C, 15 s), annealing (60°C, 30 s) and extension (72°C, 30 s) steps for 40 cycles in an Applied Biosystems™ Real-Time PCR Instruments (Life Technologies). Relative mRNA expression was calculated using the Relative Quantity (RQ) equation, RQ = 2−ΔΔCycle threshold method.
List of primer sequences for qPCR.
| Primers | Sequence |
|---|---|
| Egf | F 5′-GTCAGCTAATGGATCGAGTCA-3′ |
| R 5′-CTGCTCCCAGTTTCTACAGAAC-3′ | |
| Hb-egf | F 5′-AGCTCCGTATTCCCTCGTG-3′ |
| R 5′-CACATATGACCACACTACCGTCTTG-3′ | |
| Tgfα | F 5′-CGCCTATGGTACCTGAACATGA-3′ |
| R 5′-ACGTACCCAGAGTGGCAGAC-3′ | |
| Betacellulin | F 5′-TGAAACCAATGGCTCTCTTTG-3′ |
| R 5′-TGTCCTGGGTCTTGTGATTC-3′ | |
| P62 | F 5′-CCTTTGGCCACCTCTCTG-3′ |
| R 5′-AGGACGTGGGCTCCAGTT-3′ | |
| Fundc1 | F 5′-CCAAGACTATGAGAGCGATGAC-3′ |
| R 5′-CCGGAACTGTGGCCAAATA-3′ | |
| Bnip3 | F 5′-CAGCAATGGCAACGGTAATG-3′ |
| R 5′-CCAGAAGGATCTTCTCCATGTC-3′ | |
| Gapdh | F 5′-TGGGAAGCTGGTCATCAAC-3′ |
| R 5′-GCATCACCCCATTTGATGTT-3′ |
Immunohistochemistry
Rats were anesthetized and perfused with paraformaldehyde (PFA 4%) and picric acid (0.2%), and IHC performed following earlier protocol (Pandey et al. 2017). Briefly, rat brain was isolated, post-fixed in PFA and cryoprotected in sucrose-gradient (10, 20 and 30%). Ten micrometer coronal sections from the brain containing the hippocampus were made using cryomicrotome (Microm HM 520, Labcon, Heppenheim, Germany), mounted onto PLL-coated slides, antigen-retrieved with citrate buffer (pH 6.0), followed by 5% BSA-blocking. The hippocampal sections were probed with ERα, HB-EGF, LC3B, Beclin-1, VDAC1, cleaved caspase-3 (1:100) and NeuN (1:200) antibodies overnight and re-probed with Alexa Fluor secondary antibodies (double dilution relative to primary antibodies, 2 h). After mounting in Vectashield antifade medium containing DAPI, photomicrography of hippocampus was performed under Nikon Eclipse Ni fluorescence microscope using NIS-Elements microscope imaging software (Nikon).
TUNEL assay
Hippocampal sections were probed with TdT and fluorescein-labeled dUTP (2 h, 37°C) and immunostained with 1:200 dilution of anti-rabbit NeuN (overnight, 4°C°), as previously described (Pandey et al. 2017). The sections were mounted in Vectashield mounting medium containing DAPI and fluorescence photomicrography performed. Apoptotic index (%) was determined as the number of TUNEL-positive cells/100 nuclei, counted manually and randomly in five different areas using ImageJ 1.48v plugins.
Learning and memory tests
Passive avoidance and Y-Maze tests were performed as described earlier (Pandey et al. 2017). For passive avoidance test, rats were first subjected to acquisition in gated light-dark chambers, involving an initial acclimatization (30s) in the light compartment following by a foot shock at the dark compartment (0.5 mA, 10 s). The rats were then made to undergo shock-free retention trials of 300 s each at 24 h, 48 h and 72 h, and transfer latency time (TLT) was calculated as the time taken to shift from light to dark compartment, where increased TLT indicated better learning-memory performances. For Y-Maze test, rats were subjected to a training period of thirty trials, where they were allowed to freely move around the three arms of a Y-shaped apparatus, with one arm being shock-free and having 15-W light bulb (safe arm), while the other two arms being dark and having foot-shocks (1–5 mA) (unsafe arm). The Y-Maze memory test was performed at 24 h, 48 h and seventh day post training, and % saving memory was calculated as (Etraining−Etest) × 100/Etraining , where denoted running toward the unsafe arm.
Neuronal transfection
For siRNA silencing, 70–80% primary neurons were transfected with NT-, ERα- or Beclin-1-siRNA (100nM), as described earlier (Pandey et al. 2017). Briefly, the siRNA and Lipofectamine 2000 transfection reagent were suspended in neurobasal medium (20 min, 37°C), and the complex obtained incubated with neurons (CO2 incubator, 4–6 h). The medium was replaced with complete neurobasal medium, and study performed at 48 h post-transfection.
To examine mitophagy, 70–80% primary neurons were transfected with tandem-tagged mt-RFP-EGFP plasmid, pAT016 (0.5 μg/well of four-well chamber slide), encoding a mitochondrial-targeting signal sequence fused in-frame with RFP and EGFP genes (Kim et al. 2013, Sinha et al. 2015). The cells were co-transfected with ERα- or NT-siRNA. After 48 h, the cells were mounted in Vectashield medium containing DAPI, and fluorescence photomicrography was performed. Image analysis was conducted with ImageJ software.
MitoTracker Red staining
Primary neurons, plated on PLL-coated chamber slides, were incubated with MitoTracker Red for 30–45 min at 37°C, as described earlier (Kaplan et al. 2012). The cells were permeabilized with PBST (PBS + 0.1% Triton X 100), mounted in Vectashield medium containing DAPI, and fluorescence photomicrography was performed.
Mitochondrial respiration and ATP production measurement
Primary hippocampal neurons were plated as 40,000 cells/well onto the PLL-coated Sea-horse V7-PS Flux 24-well polystyrene 24-well plates. The cells were grown for 5 days in neurobasal medium supplemented with N2 (1%), B-27 (2%) and l-glutamine (2 mM), and then incubated with NT-si RNA (100 nM), ERα-si RNA (100 nM), NT-siRNA + HB-EGF (5 ng/mL) or ERα si RNA + HB-EGF for 48 h. The cells were washed with PBS and XF Base minimal DMEM (Agilent Technologies), used as mitochondrial respiration assay medium. The mitochondrial respiration was assayed by stepwise addition of A: oligomycin (1 μM), B: carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (2 μM) and C: rotenone (0.5 μM). Three measurement cycles of a 2-min mix, 1-min wait, and 5-min measure were performed after each addition, using the Sea-horse Bioscience XFe-24 analyzer (Agilent Technologies) (Pal et al. 2019).
Statistics
For statistical analyses, GraphPad Prism (GraphPad Software) was used. Unpaired Student t-test with two-tailed statistics were conducted for comparison between two groups of independent samples. For comparison between more than two groups, one-way ANOVA was performed, followed by the Tukey’s post hoc multiple comparisons test. Two-way ANOVA followed by Tukey’s post hoc multiple comparisons test was performed for comparisons between more than two groups and more than one parameter.
Results
Effect of OVX on hippocampal autophagy
We generated OVX female rats and investigated hippocampal autophagy in the rats. We detected a significantly increased LC3-II level at day 21 post OVX surgery (Fig. 1A), and hence, detailed experiments were performed at this time point. A reduced serum level verified estrogen deficiency in the OVX rats (Table 2).
Serum estradiol levels in rats.
| Groups | Estradiol (pg/mL) |
|---|---|
| Sham | 94.88 ± 8.24 |
| OVX | 36.46 ± 5.65a |
| OVX+E2 | 84.07 ± 7.19b |
Data represent means ± s.e. of three rats/group.
aP < 0.001 and bP < 0.001 compared to Sham and OVX respectively.
Along with LC3-II, we observed increased levels of autophagy markers, Beclin-1, ATG-7 and ATG5/12 conjugate, in the hippocampus of OVX rats compared to sham (Fig. 1B). We then examined whether the serum estrogen deficiency could be responsible for this increased hippocampal autophagy and found that E2 supplementation, which restored serum E2 levels (Table 2), reduced the OVX-induced changes in hippocampal autophagy markers (Fig. 1B). To determine hippocampal autophagic flux in OVX rats, we measured P62 (also known as Sequestosome-1 (SQSTM1)), which undergoes autolysosomal degradation during autophagy (Bjorkoy et al. 2009). We observed a decrease in hippocampal p62 levels in the OVX rats and its recovery by E2 (Fig. 1B). Nonetheless, measuring the hippocampal P62 mRNA showed its up-regulated expression in OVX rats (Fig. 1C). Thus, our results demonstrate that despite increased P62 transcription, its protein levels were significantly reduced, indicating an OVX-induced enhanced autophagic flux. We further explored the autophagy regulators, and detected an OVX-mediated decrease in p-AKT/AKT, p-mTOR/mTOR and p-ULK1 (S757)/ULK1 in the hippocampus, and their E2-mediated recovery (Fig. 1D), suggesting an induction and initiation of hippocampal autophagy in OVX condition. Our TEM data demonstrating enhanced OVX-mediated hippocampal accumulation of autophagosome and its E2-mediated reduction (Fig. 1E) validated the increased OVX-induced autophagy.
Effect of OVX on hippocampal EGFR signaling and its link with autophagy
EGFR signaling often regulates the cellular AKT pathway (Jin et al. 2005), and given our results showing OVX-mediated reduction in hippocampal p-AKT/p-mTOR levels, we asked if EGFR signaling participated in the OVX-induced effects. For this, we first checked the effects of OVX on hippocampal EGFR signaling. We detected an OVX-mediated reduction in hippocampal p-EGFR, indicating decreased EGFR activation, which could be inhibited by E2 treatment (Fig. 2A). We next screened the EGFR ligands through qPCR, and identified an OVX-mediated decrease in hippocampal HB-EGF mRNA, while the other ligands were unaffected (Fig. 2B). The Western blotting data validated the OVX-induced HB-EGF reduction and recovery by E2 treatment (Fig. 2C). To prove the HB-EGF and EGFR link, we administered HB-EGF in the hippocampus of OVX rats. We observed an HB-EGF-mediated recovery in p-EGFR levels of OVX rats, comparable to that in the OVX + E2 group (Fig. 2D), overall, suggesting that hippocampal HB-EGF/EGFR signaling was E2 dependent.
OVX downregulates hippocampal HB-EGF/EGFR signaling. Hippocampal tissues from Sham, OVX, OVX+E2− or OVX+HB-EGF-treated rats were isolated. (A, C and D) Representative Western blots and densitometry of p-EGFR (A and D) and HB-EGF (C) normalized with EGFR (A and D) and β-actin (C). (B) qPCR analysis showing mRNA levels of EGFR ligands normalized with housekeeping gene, Gapdh. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
We then investigated whether the attenuated hippocampal HB-EGF/EGFR signaling in the OVX rats was linked to autophagy. We first examined the effect of intra-hippocampal HB-EGF administration on AKT-mTOR pathway, and observed that hippocampal levels of p-AKT, p-mTOR and p-ULK1 (Ser757) in OVX rats were restored to that achieved by the E2 administered group (Fig. 3A). Besides reduction in AKT-mTOR signaling, we also observed increased AMPKα activation in OVX rat hippocampus, and this effect was restored via HB-EGF and E2 treatment (Fig. 3A). Furthermore, the OVX-induced changes in hippocampal LC3-II, Beclin-1, ATG-7 and p62 levels (Fig. 3B) were reversed upon HB-EGF treatment in the OVX rats, on a par with E2-treated group. Thus, our data indicate that OVX causes an increased hippocampal autophagy via reduced HB-EGF/EGFR signaling.
OVX induces hippocampal autophagy via decreased HB-EGF/EGFR signaling. Hippocampal tissues from Sham, OVX, OVX+HB-EGF− and OVX+E2-treated rats were isolated. (A and B) Representative Western blots and densitometry of autophagy regulators (A) and the autophagy markers (B) normalized with respective non-phospho counterparts (A) and β-actin (B). Data represent means ± s.e. of three rats/group. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to sham or as indicated.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Role of ERα and effect of OVX on HB-EGF/EGFR and autophagy in hippocampal neurons
We validated our findings through in vitro studies in primary hippocampal neurons (that regulates cognitive functions). We used ERα-silenced primary hippocampal neurons, following our in vivo observation showing reduced ERα in the NeuN-expressing cells of the hippocampus (ERβ levels remained unchanged, data not shown) of OVX rats and its recovery by E2 treatment (Fig. 4A). Efficacy of ERα silencing has been shown in Fig. 4B. In line with our in vivo findings (Figs 1, 2 and 3), we observed an ERα-siRNA-mediated reduction in HB-EGF (Fig. 4C), p-EGFR, p-AKT/AKT, p-mTOR/mTOR and p-ULK1/ULK1 levels (Fig. 4D), along with a concomitant increase in LC3-II, Beclin-1, ATG-7 and ATG5/12 and decrease in p62 expression levels in the primary hippocampal neurons (Fig. 4E). The ERα-siRNA-mediated changes could be blocked by HB-EGF (Fig. 4D and E).
ERα depletion suppresses HB-EGF/EGFR signaling, inducing autophagy in hippocampal neurons. Hippocampal sections were made from Sham, OVX and OVX+E2-treated rats and immunofluorescence performed. (A) Representative fluorescence photomicrograph (20×) and quantification relative to sham of ERα and NeuN co-immunostaining and nuclear DAPI counter-staining. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. Rat primary hippocampal neurons were transfected with NT-siRNA or ERα-siRNA. (B and C) Representative Western blot and densitometry of ERα (B) and HB-EGF (C) normalized with β-actin. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF. (D and E) Representative Western blot and densitometry of p-EGFR (D), autophagy regulators (D) and autophagy markers (E) normalized with respective non-phospho counterparts (D) and β-actin (E). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to NT-siRNA or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
We verified the above observations in OVX rats, which showed decreased HB-EGF (Fig. 5A) and increased autophagy (Fig. 5B and C) in the NeuN-expressing matured neurons in the hippocampus. Thus, our data indicate an ERα downregulation, which in turn induces increased autophagy in the hippocampal neurons via reduced HB-EGF/EGFR signaling.
OVX reduces HB-EGF and induces autophagy in the neurons of hippocampus, which are suppressed by HB-EGF or E2 treatment. Hippocampal sections were made from Sham, OVX, OVX+HB-EGF- or OVX+E2-treated rats and immunofluorescence performed. (A, B and C) Representative fluorescence photomicrographs (20×) and quantification relative to sham of HB-EGF (A), LC3 (B) and Beclin-1 (C) co-immunolabeled with NeuN and counter-stained with nuclear DAPI. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Role of altered HB EGF and autophagy in OVX and ERα- siRNA -induced hippocampal neuronal apoptosis
We next examined whether the reduced HB-EGF and augmented autophagy participated in OVX-induced hippocampal neuronal apoptosis, reported earlier (Sales et al. 2010, Yazgan & Naziroglu 2017). By treating HB-EGF or the autophagy inhibitor, 3-MA (of note, the efficacy of 3-MA as an autophagy inhibitor in vivo was verified by analyzing LC3-II levels (Fig. 6A)), we found a reduced neuronal cleaved caspase-3 expression (Fig. 6B) and apoptotic index (%) (by TUNEL assay) (Fig. 6C), at par with E2-treated group, in the hippocampus of OVX rats. Thus, our data indicate an attenuated HB-EGF/EGFR and increased autophagy-mediated neuronal apoptosis in the hippocampus of OVX rats. A reduced hippocampal NeuN level, suggesting neuronal loss, in the OVX rats, and its HB-EGF and 3-MA-mediated recovery (Fig. 6B, C and D) supported the apoptosis concept. Further, our in vitro data validated the in vivo findings, showing decreased cleaved caspase-3 and cleaved PARP/PARP levels following HB-EGF treatment or autophagy inhibition using Beclin-siRNA (of note, the efficacy of Beclin-siRNA was verified (Fig. 6E)) in ERα-siRNA-treated primary hippocampal neurons (Fig. 6F).
OVX and ERα-silencing induces hippocampal neuronal apoptosis via reduced HB-EGF and increased autophagy. Western blotting and IHC were performed on hippocampal tissues and sections of Sham, OVX, OVX+3-MA, OVX+HB-EGF− or OVX+E2-treated rats. (A and D) Representative Western blot and densitometry of LC3-II (A) and NeuN (D) normalized with β-actin. (B and C) Representative fluorescence photomicrographs (20× magnification) and quantification relative to sham of cleaved caspase-3 (c-Cas 3) and NeuN co-immunostaining (B), TUNEL and NeuN antibody co-staining (C), with nuclear DAPI counter-staining (B and C) in the rat hippocampal sections. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF or co-transfected with Beclin-siRNA. (E and F) Representative Western blot and densitometry of Beclin-1 (E), cleaved caspase-3 (c-Cas 3) and cleaved PARP/PARP (F) normalized with β-actin. Data represent means ± s.e. of three independent primary neuron culture preparations. ***P < 0.001 and **P < 0.01 compared to NT-siRNA or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
OVX-mediated apoptosis: possible involvement of excessive mitophagy
We investigated the basis of hippocampal autophagy-mediated apoptosis in OVX rats. Based on our results showing increased activation of AMPKα, possibly suggesting ATP deficit in OVX hippocampus, we examined mitophagy, the process of excessive targeting of mitochondria by selective autophagy, earlier reported to induce neuronal apoptosis under different conditions (Ha et al. 2017, Feng et al. 2018). To test this, we assessed the VDAC1 and COXIV protein levels in the hippocampus, and observed their reduction in OVX group compared to sham, and a 3-MA (autophagy inhibitor)-mediated recovery (Fig. 7A). Thus, our results indicate an autophagy-dependent mitochondrial loss in the hippocampus of OVX rats. We related the OVX-induced mitochondrial loss with HB-EGF and E2 and observed a recovery in VDAC1 and COXIV protein levels (Fig. 7B) and mitochondrial CytB and COXII genes (Fig. 7C) following HB-EGF and E2 treatment. We further observed an OVX-mediated reduction in VDAC1 expression in NeuN-expressing cells of the hippocampus, and its E2, HB-EGF and 3-MA-mediated recovery (Fig. 7D).
OVX induces mitochondrial loss in hippocampal neurons, which is restored by 3-MA, HB-EGF or E2 treatments. Hippocampal tissues were isolated for Western blotting and mitochondrial DNA content measurement, and hippocampal sections were made from Sham, OVX, OVX+3-MA, OVX+HB-EGF− or OVX+E2-treated rats. (A, B and E) Representative Western blots and densitometry of mitochondrial markers normalized with β-actin (A and B) and mitochondrial PINK1, Parkin and LC3-II levels normalized with VDAC1 (E). (C and F) qPCR analysis shows relative mtDNA (CytB and COXII) normalized with nuclear DNA (18S rRNA gene) (C) and Fundc1 and Bnip3 normalized with Gapdh (F). (D) Representative fluorescence photomicrographs (20×) and quantification relative to sham of VDAC1 co-immunolabeled with NeuN and counter-stained with nuclear DAPI. Scale bar: 100 μm. Data represent means ± s.e. of three rats/group. ***P < 0.001 and **P < 0.01 compared to sham or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
We next investigated mitophagic mediators, PINK1/Parkin, Bnip3 and Fundc1 (Amadoro et al. 2014, Cummins & Gotz 2018, Lampert et al. 2019) at protein or mRNA levels in the hippocampus of OVX rats. We did not find significant changes in mitochondrial PINK1 and Parkin levels in the hippocampus during OVX-mediated mitophagy from that observed under basal condition (Sham), despite significant enrichment of LC3-II in the mitochondrial fraction (that indicates mitophagy) and its E2-mediated reduction (Fig. 7E). Similarly, we also observed an unaltered expression of Bnip3 and Fundc1 (Fig. 7F), known to participate in mitophagy (Liu et al. 2014), in the OVX rat hippocampus. Hence, mitophagy mediators, other than PINK/Parkin, Bnip3 and Fundc1 further need to be investigated for understanding mechanisms of OVX-induced hippocampal mitochondrial loss.
Effect of ERα-silencing on mitophagy and ATP production in primary hippocampal neurons
We further explored mitophagy in ERα-silenced primary hippocampal neurons, using a tandem-tagged mtRFP-EGFP chimeric plasmid that relies on different stabilities of RFP and GFP in an acidic environment (Kim et al. 2013, Sinha et al. 2015). We observed that in NT-siRNA-transfected cells, both EGFP and RFP signals co-localized (yellow: red + green, indicating mitochondrial presence in cytosol), whereas the knockdown of ERα increased RFP signal alone, indicating autolysosomal-resident mitochondria where EGFP signal was quenched at an acidic pH, suggesting mitophagy (Fig. 8A). We further verified the reduction in mitochondrial content in ERα-silenced primary hippocampal neurons using MitoTracker Red dye (Fig. 8B). An HB-EGF-mediated reduction in mitophagy and recovery in mitochondrial content in the primary hippocampal neurons (Fig. 8A and B) suggested a protective role of the growth factor against autophagy-induced mitochondrial loss in ERα-suppressed condition. Furthermore, we also observed an HB-EGF-mediated rescue from excessive ERα-siRNA-induced loss in ATP production, using Sea-horse Bioscience XFe-24 analyzer (Fig. 8C).
ERα depletion induces mitophagy, mitochondrial loss and reduced ATP production in primary hippocampal neurons, which are reduced by HB-EGF treatment. NT-siRNA or ERα-siRNA was transfected in rat primary hippocampal neurons, with/without HB-EGF. (A) Representative fluorescence photomicrograph (60×) and quantitative analysis of cells transfected with tandem-tagged mt-RFP-EGFP plasmid. Fluorescence signals indicate the expression of mt-RFP-EGFP targeting mitochondria: yellow color, no mitophagy or cytosolic mitochondria; red color, mitophagy or mitochondria inside lysosomes. Scale bar: 20 μm. Transfected cells from three-four random fields were taken for analysis. (B) Representative fluorescence photomicrograph (60×) and quantification of Mitotracker and nuclear DAPI co-staining. Enlarged: Inset area of merged image. Scale bar: 20 μm. Cells from three-four random fields were taken for analysis. (C) Sea-horse analysis of ATP production (in fold change). Data represent means ± s.e. of three independent primary neuron culture preparations. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to NT-siRNA or as indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Effect of HB-EGF, 3-MA and E2 on OVX-induced cognitive impairment
We finally linked our observations of reduced hippocampal neuronal HB-EGF and increased autophagy with learning-memory performances in OVX rats. We found that while OVX caused a reduction in TLT in passive avoidance test (Fig. 9A) and increased error (%) and decreased saving memory (%) in Y-Maze test (Fig. 9B), HB-EGF and 3-MA could inhibit these changes. The effects of HB-EGF and 3-MA were comparable to that of OVX+E2 (Fig. 9A and B), overall indicating an attenuated HB-EGF/EGFR and increased autophagy-dependent leaning-memory dysfunction in OVX rats.
Reduced HB-EGF and increased autophagy induce cognitive deficits in OVX rats. Passive avoidance and Y-Maze tests for learning-memory performances were carried out with Sham, OVX, OVX+HB-EGF−, OVX+3-MA− or OVX+ E2-treated rats. (A) Representative bar graphs depicting transfer latency time (TLT) in passive avoidance test. Data represent means ± s.e. of eight rats/group. ###P < 0.001 compared to acquisition trial. ***P < 0.001 and $$P < 0.01 compared to Sham and OVX for a particular retention trial respectively. (B) Representative bar graphs depicting % number of errors and % saving memory assessed at 24, 48 h and 7 days post-learning in Y-maze test. Data represent means ± s.e. of seven rats/group. ***P < 0.001 and **P < 0.01 compared to sham; $$P < 0.01 and $P < 0.05 compared to OVX.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Discussion
The current study reveals a novel mechanism of hippocampal neuronal damage and cognitive dysfunction in adult females caused by ovariectomy. Through the study, we propose a mechanism of OVX-induced ERα deficiency and HB-EGF/EGFR inactivation, triggering a reduced p-AKT/mTOR-mediated increased autophagy and mitochondrial loss, ultimately leading to hippocampal neuronal apoptosis and learning-memory impairment (Fig. 10).
Schematic proposing deregulated HB-EGF/EGFR-dependent autophagy, mitochondrial loss, hippocampal neuronal apoptosis and cognitive deficits in E2/ERα deficiency. E2 deficiency attenuates ERα and then suppresses HB-EGF/-dependent EGFR activation in hippocampal neurons. Reduced HB-EGF/EGFR signaling further down-regulates AKT/mTOR/ULK1 pathway of autophagy regulation, resulting in increased LC3/Beclin and decreased p62-mediated autophagy, autophagosome formation and mitochondrial loss. This deregulated HB-EGF/EGFR and autophagy pathway forms a key reason for hippocampal neuronal apoptosis and learning-memory impairment in OVX rats, which may be inhibited by E2 or HB-EGF supplementation. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0197.
Citation: Journal of Endocrinology 244, 1; 10.1530/JOE-19-0197
Estrogen prevents aberrant hippocampal neuronal apoptosis and cognitive deficits (Sales et al. 2010, Uzum et al. 2016, Yazgan & Naziroglu 2017), and the present study establishes the contribution of a controlled autophagy mechanism in estrogen-mediated neuroprotection. Here, we essentially demonstrated that estrogen deficiency up-regulates the expression of autophagy proteins such as Beclin1, LC3 and ATG7 along with the levels of ATG5/ATG12 conjugate within the hippocampus, indicative of an estrogen-regulated phagophore elongation and association with theautophagic vesicles. We then explored the regulatory pathway of autophagy, and our results pointed to the nodal role of ULK complex in hippocampal neuronal survival, where estrogen-mediated responses extended to the autophagy machinery via a coordinated balance with AKT/mTOR. Consistent with our observations, earlier reports showed an estrogen deficiency-induced autophagy in fibroblasts, osteocyte-like cell line, differentiating osteoclasts and blastocysts in the uterus, where 17β-estradiol treatment or inhibition of autophagy reversed skeletal, bone and uterine damage and reduced obesity via LC3-II and mTOR-related signaling cascades (Choi et al. 2014, Lin et al. 2016, Leu et al. 2017, Fu et al. 2018, Tan et al. 2018). Conversely, increased autophagy helped maintaining the normal functions of alveolar bone osteocytes (Florencio-Silva et al. 2018), osteoblasts derived from elderly females (Camuzard et al. 2016), aged female heart (Garvin et al. 2017) and bone marrow mesenchymal stem cells (Qi et al. 2017) following OVX. Likewise, autophagy contributed to the estradiol-mediated protective effect on endotoxemia-induced multiple organ dysfunction (Chung et al. 2017), overall indicating a cell type specific effect of estrogen deficiency on autophagy. In terms of the brain, there have been two in vivo reports, one demonstrating a short- and long-term ovarian hormone-dependent differential response on autophagy in the cortex-hippocampus mixture (Yao et al. 2018), and the other claiming increased hippocampal autophagy following OVX (Fang et al. 2018), marked by altered p62, Beclin-1, ATG5 and LC3-II protein levels. However, unlike ours, the studies did not delve into the autophagy regulation mechanism particularly in the hippocampal neurons. The current study provided a detailed understanding on ovariectomy-induced autophagy using suitable modulators, and also offered a growth factor regulated mechanism supporting the concept. Our study further extended the observations in terms of mitophagy, hippocampal neuronal apoptosis and neuronal loss, culminating in cognitive decline.
Our data for the first time add HB-EGF/EGFR to the list of growth factor signaling mechanisms, viz. nerve growth factor/tropomyosin receptor kinase A (TrkA) (Sarvari et al. 2017, Liu et al. 2018), brain-derived neurotrophic factor/TrkB (Murphy et al. 1998) and insulin-like growth factor (Witty et al. 2013), controlling estrogen-induced responses to hippocampal neurons. Additionally, although an altered EGFR signaling is well-reported in relation to autophagy for various cell types and conditions (Graham et al. 2016, Wang et al. 2017), our study emerged first in linking HB-EGF ligand with autophagy, thereby, opening a new direction to HB-EGF-induced signaling mechanisms within the hippocampus. Hence, for both E2-dependent and -independent conditions, mitochondrial dynamics presumably regulates the typical functions of HB-EGF, viz., survival, mitogenesis, anti-excitotoxicity, and so forth, particularly in hippocampal neurons. Likewise, for amyloid beta toxicity, aging, learning memory impairments (Shariatpanahi et al. 2016, Huang et al. 2018), and so forth marked by hippocampal autophagy, an intermediate involvement of HB-EGF seems worth exploring.
Cross-talk between E2 and EGFR has been mainly reported in triple-negative breast cancer, and is characterized by dysregulated PI3K/AKT/mTOR signaling (Araki & Miyoshi 2018). Irrespective of estrogen treatment, an EGFR, PI3K/AKT/mTOR and autophagy link is also known, primarily in cancerous cell, such as, hepatic cancer, glioblastoma, and so forth (Palumbo et al. 2014, Dai et al. 2018). Our data reflect a combination of all these pathways, demonstrating the regulatory role of hippocampal neuronal AKT/mTOR at the cross-road of E2, EGFR and downstream autophagy, rarely explored in the non-malignant cells. Interestingly, our study draws support from an earlier concept (Shafi 2016) hypothesizing an inverse relation between cancer and Alzheimer’s Disease-induced hippocampal neurodegeneration, where the former generally involves an upregulated EGFR-PI3K/AKT/mTOR signaling and reduced autophagy as opposed to the latter. However, analyzing our results indicate that the reduction in HB-EGF/EGFR activation, following estrogen deficiency, suppresses the membrane recruitment of cytoplasmic PI3K and subsequent phoshatidylinositol-3,4,5-trisphosphate and AKT-dependent mTOR activation. Thus, taken together, although estrogen receptor itself appears capable of activating PI3K (Toss & Cristofanilli 2015), our findings reveal the essential involvement of an activated EGFR or rather cross-talk between EGFR and estrogen receptor for recruiting PI3K/AKT/mTOR and sustaining mitochondrial homeostasis and an ultimate hippocampal neuronal survival. Nonetheless, detailed future studies are necessary to understand the interactions of estrogen receptor and HB-EGF/EGFR signaling toward regulating the complex association between autophagy and mitochondria. Our study further addressed in details the relationship between estrogen deficiency, autophagy and neuronal apoptosis within the hippocampus through in vivo experiments, and then validated the process in ERα-deficient cultured hippocampal neurons. Secondly, through in vivo and in vitro methods using appropriate autophagy inhibitors, our results demonstrated a direct autophagy-dependent apoptosis within hippocampal neurons during estrogen or ERα-deficiency, and further associated this process with cognitive deficits. All these events were blocked by E2, thereby defining novel mechanisms by which this hormone affords protection against hippocampal neurodegeneration. Moreover, an ERα antagonist or EGFR inhibitor-mediated increase in LC3-II and Beclin-1 expression levels in the primary hippocampal neurons (data not shown) supported that E2 deficiency-mediated ERα reduction and EGFR inactivation induce autophagy in the hippocampal neurons.
Although autophagy is generally neuroprotective, been reported for memory formation (Glatigny et al. 2019) and as a survival mechanism in dementia (Hu et al. 2017, Lee et al. 2017) its overactivation in neurons has also been known to induce neurodegeneration. In hypoxic ischemic brain injury, autophagy gene-deletion significantly prevented severe hippocampal damage in neonatal or adult mice (Koike et al. 2008). Similarly, beneficial effects of autophagic inhibition against neuronal cell death have also been demonstrated in a 6-hydroxydopamine model of substantia nigral injury, which exhibits increased autophagy (Li et al. 2011). Additionally, neuronal death elicited by dysfunctional ESCRT-III, which is associated with frontotemporal dementia linked to chromosome 3, could be rescued by both pharmacological and genetic inhibition of autophagy, suggesting a pro-death function of neuronal autophagy (Lee & Gao 2009). Interestingly, our results suggest that increased mitophagy could be the mechanism how estrogen deficiency-induced autophagy leads to neuronal apoptosis and loss. Mitochondrial activity is necessary for both neuronal function and its survival wherein, mitochondrial pruning for removing damaged mitochondria is essential to sustain mitochondrial health in cells, including neurons (Sinha et al. 2015). This selective degradation of malfunctioning mitochondria is executed by mitophagy, defined as an autophagy-dependent mitochondrial degradation (Sinha et al. 2015). Intriguingly, mitophagy, like autophagy, is not always protective, as increased mitophagy may also lead to increased neuronal loss (Chakrabarti et al. 2009, Wong & Cuervo 2010, Shi et al. 2014, Ha et al. 2017, Feng et al. 2018). In line with these reports, our results also support the notion that increased mitochondrial loss under E2/ERα deficiency may result in energy crisis, leading to enhanced apoptosis. However, since mitophagy involves membrane depolarization and different mitophagy mediators (Grenier et al. 2013), the participation of these mitophagy pathways in E2 deficiency-induced hippocampal neuronal apoptosis and cognitive impairment still need to be explored. Hence, studies are currently underway toward a detailed understanding of the mechanistic basis of OVX-induced mitophagy, and especially the role of mitophagy proteins during mitochondrial clearance in the hippocampal neurons. Particularly, although OVX failed to impact PINK/Parkin (mitochondrial), BniP3 and Fundc1 expression levels in hippocampus, the likelihood of their functional involvement in ERα-deficient hippocampal neurons also appears worth probing.
In conclusion, the current study showed that E2 governs HB-EGF/EGFR-dependent autophagy and mitochondrial integrity in hippocampal neurons, and this event may be disrupted in menopause triggering hippocampal neurodegeneration. Overall, our study reveals intersection points of HB-EGF, autophagy, mitochondrial loss and apoptosis as key players in E2-mediated hippocampal neuroprotection, which may be pharmacologically targeted for restricting cognitive dysfunction, particularly in elderly women.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Science and Engineering Research Board, Govt. of India [GAP340].
Author contribution statement
R P contributed to the experimental plan, design, performance (animal treatment, majority Western blotting, IHC, ATP assay and neurobehavioral assay), data analysis and paper writing. P S contributed to the animal treatment and Western blotting. B A contributed to Western blotting of all ATG5/12 and Fig. 7B and E. H P G contributed to qPCR, statistical analyses and data analyses. S P contributed to mitochondrial DNA content and ATP-based experiment. N A carried out TEM. K G contributed to animal treatment and neurobehavioral assay. N C contributed to experimental designing and supervision. R A S contributed to the planning, data analyses and paper writing for all mitochondria-associated experiments. S B contributed to the overall experimental planning, designing, supervision and paper writing.
Acknowledgement
R P was supported by UGC fellowship, Govt. of India. Juhi Mishra (SRF, CSIR-IITR) and Rafat Malik (P. A., SERB) provided suggestions following thorough reading of the manuscript. CSIR-IITR manuscript number is 3573.
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